Streptavidin coupled alkaline phosphatase

96-well plates (Nunc) were coated overnight at 4°C with 5 μg/ml anti-IFN-γ capture antibody (BioLegend—clone R4-6A2) and blocked with 2.5% FCS (Life Technologies) in PBS. Subsequently, the cell supernatant and a known IFN-γ (PeproTech) concentration were added. After 4 h the plate was washed with PBS containing 0.005% Tween-20 followed by incubation with a biotinylated anti-IFN-γ detection antibody (Biolegend—clone XMG1.2) for 1 h. Wells were washed and subsequently incubated for 1 h with streptavidin-coupled alkaline phosphatase (GE Healthcare). For detection the substrate solution SIGMAFAST p-Nitrophenyl phosphate (Sigma-Aldrich) was added. After stopping the reaction with 3 M NaOH the absorbance was measured at 405 nm.

To determine gp120 and p24 content of virus preparations, env-pv^NLLuc viruses were produced in 293-T cells (T25 flasks; 750.000 cells in 5 ml medium, seeded 24 h pre-transfection). The medium was changed 12 h post-transfection and virus-containing supernatants harvested 48 h post-transfection. The supernatants were cleared by low speed centrifugation (300 g, 3’), then ultracentrifuged (SW28 rotor, 2 h, 28.000 rpm, 4°C), the supernatant removed and viral pellets resuspended in 0.3 ml cold PBS and stored at −80°C. Virion associated p24 and gp120 antigens were quantified by ELISA as previously described [13 (link)]. Briefly, virus preparations were dissolved in 1% Empigen (Fluka Analytical, Buchs, Switzerland) and dilutions of each sample probed for gp120 and p24. Gp120 was captured on anti-gp120 D7324 (Aalto Bioreagents, Dublin, Ireland) coated immunosorbent plates and detected with biotinylated CD4-IgG2 and Streptavidin-coupled Alkaline Phosphatase (GE Healthcare, Chalfont St Giles, UK). P24 was captured on anti-gp120 D7320 (Aalto Bioreagents, Dublin, Ireland) coated plated and detected using Alkaline Phosphatase-coupled antibody BC1071-AP (Aalto Bioreagents, Dublin, Ireland).